Genetic analysis
of CSFV E2 gene was done for total 80 Indian isolates collected from pigs of
different States. It was found that 20 viruses belonged to genotype 1.1 and 3
viruses belongs to genotype 1.2. The isolate no.CSF/MZ/KOL/73, CSF/MZ/SAI/76, CSF/MZ/AIZ/87
and AIZAWL-09(CSF/MZ/AIZ/115) isolated from domestic pigs in Mizoram are
grouped in genotype 1 and subgenotype 1.1. The AIZAWL-09(CSF/MZ/AIZ/115),
isolated from Zovawk (semi wild local pigs of Mizoram ) showed 99% homology
with the  HCLV, Lapinized vaccine strain
and 96% homology with Alfort A19, Chinese C strain, Alfort/187 and
Weybridge  strains and grouped separately
from other three isolates from Mizoram. Isolate CSF-MZ-AIZ-352
and CSF-MZ-AIZ-348 also showed 99% homology with HCLV-India (Indian vaccine).
Isolate no CSF/MZ/KOL/73 and CSF/MZ/SAI/87 isolated from domestic crossbred
pigs shared 99% homology with Assam isolate from pigmy hog (EU567078) and
grouped together but away from the isolate no AIZAWL-09(CSF/MZ/AIZ/115). These
isolates from NE States also belong to genotype 1.1. Isolate from Maharastra
also belongs to genotype 1.1. CSFVPK15C-NG79-11,
CSFV-IVRI-VB-131, BHD03, CSF212L-13 belongs to genotype 1.1. Total 17 Indian
isolates fell under genotype 2.1 out of which two Indian isolates from
the northeast detected during 2002 and 2006, belonged to genotype 2.1. The
isolate CSF/MZ/LUN/421 and CSF/MZ/CHM/425 isolated from two different outbreak
of CSF in wild pigs (Sus Scrofa) of
Mizoram shared 98% homology and grouped in the genotype 2 and subgenotype of
2.1 along with two other isolates LOKHORA 169 and CSF-40-02 detected in
domestic pigs from Assam. CSF/MZ/LUN/421, isolated from wild pigs in Mizoram
shared 99% homology with LOKHORA 169, isolated from domestic pigs in Assam.
Whereas CSF/MZ/LUN/425, isolated from wild pigs in Mizoram shared 97% homology
with LOKHORA 169. Recently sequenced isolate IND-AS-GHY-G4, AS-7-K, AS-2-K and
AS-6-K collected from Assam grouped in genotype 2.1.  All the group 2.1 isolates appeared closely
linked, clustered together, indicating circulation of similar viruses in pigs
of Assam. These viruses were closely related to some isolates circulating in
China (GXW02) and Taiwan (0406/CH/01/TWN). Both the isolates CSF/MZ/LUN/421 and
CSF/MZ/CHM/425 shared 91-94% homology with these Chinese isolates, indicating
possible close links or source of origin. Total 48 Indian isolates of CSFV were
typed as genotype 2.2 and these were collected between years 2006 to 2015. All
Indian isolates clustered very closely and had highest similarity with Strain
39 and ZJ-2/99 from China which were first detected in the year 1999. When the
complete E2 gene sequence (1140 nts) of CSFV/UP-BR-793/09 was blasted, it
revealed maximum homology (96%) with Strain-39 which has putatively arose due to
natural recombination between Sheimen and GXGW02. But the Indian isolate,
CSFV/UP-BR-757/09 does not share close homology with either Strain-39 or its
parent virus in the 5’UTR region.KHG-06
showed 99% homology with Indian viruses and 97% with isolate CSF1060 from Nepal
collected in 2011. CSFV-UP-BD-BD-11 also showed 97% homology with CSF1059 from
Nepal collected in 2011. All the 61 isolates however showed distant homology
with the Indian vaccine strain with homologies ranging from 88.7 to 89.5%; the
vaccine strain belongs to genogroup 1.1.

Phylogenetic analysis on the basis of
NS5B sequence comparison

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Phylogenetic analysis based on NS5B gene sequences of 56 Indian isolates was conducted and the results corroborated those obtained in E2 and 5’NTR gene analysis. Out of 56 Indian isolates 20 fell in 1.1 3 in 2.1 and majority 33 fell in genotype 2.2.Isolate TUCN collected from Tamilnadu belonged to genotype 2.2. The isolate CSF/MZ/LUN/421 and CSF/MZ/CHM/425 grouped into genotype 2.1 as in E2 analysis. Another isolate from Tamilnadu (TNVLI) and NE States belongs to genotype 1.1. Isolate collected from Dehradun and Lalkuan from Uttarakhand belonged to genotype 2.2. Strain 39 distributed in two different genogroup 1.1 and 2.2. 5´UTR and NS5B genes based tree clustered Strain 39 into subgroup 1.1 while E2 gene based tree clustered into subgroup 2.2. Hence Strain 39 virus must contain the genetic variability or recombination of two different CSFV genogroup viruses. Our study revealed that majority of Indian isolates belongs to genotype 1.1 and 2.2.

Phylogenetic analysis on the basis of
complete genome sequence comparison

Total 98
isolates with complete genome included in the study (table 12). Phylogenetic
analysis based on complete genome revealed that NG79-11 and CSFV212L-13 belongs
to genotype 1.1 and ND-169-11, SKN-11, NVD-11, KHG-06 isolate sequenced in this
study in either of the phylogenetic trees were fall under subgroup 2.2 of
genogroup 2 along with earlier published Indian CSFV isolates and other
reference isolate stran 39. The isolates ND-169-11, SKN-11, NVD-11, and KHG-06
are closely related to Strain 39 virus. NG79-11 and other recently sequenced
Indian isolates CSFV-IVRI-VB-131 shared very close homology. Phylogenetic
analysis segregated virulent, moderately virulent and low virulent or vaccine
strains into distinct clusters. Most of the virulent and vaccine strains
clustered into same group or genotype i.e. genotype 1 while moderately virulent
strains clustered into genotype 2 and 3. Phylogenetic analysis conducted in
this study proves the presence of genotype 1 and 2 in India. In conclusion most
of the recent Indian isolates from different parts of India are from both
genotypes (1& 2) viruses. Genotype 1 is circulating mainly in NE states of
India whereas the genotype 2 is predominantly circulating in all over India.
Genotype 2.2 is dominantly circulating in all over India.